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Thermostable Transketolase from

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Abstract

Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTKgst) retained 100% activity for one week at 50 °C and for 3 days at 65 °C, and has an optimum temperature range around 60–70 °C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one‐step purification by heat shock treatment of crude cell extracts at 65 °C for 45 min, directly yielding 132 mg of pure mTKgst from 1 L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 °C than at 20 °C, and 4 times higher at 50 °C when compared to E. coli TK under identical conditions. When tested at 50 °C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 °C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors – glycolaldehyde, D‐glyceraldehyde and butyraldehyde – from which the corresponding products L‐erythrulose 1, D‐xylulose 2 and 1,3‐dihydroxyhexan‐2‐one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non‐hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non‐conventional media.
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Document Type: Research Article

Affiliations: 1: -64287 Darmstadt, Germany 2: CEA, DSV, IG, Genoscope, 2 rue Gaston-Crémieux, F-91057 Evry, France 3: Isthmus SARL, 81 rue Réaumur, F-75002 Paris, France

Publication date: 14 January 2013

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